摘要
为了探讨人肥胖（obesity，OB）基因的生理和病理意义，利用逆转录-聚合酶链式．反应（RT-PCR）方法，从中国汉族成人腹膜后脂肪组织总RNA中扩增出肥胖基因编码区序列（cDNA）．定向亚克隆pUC19质粒，克隆的OBcDNA不含信号肽序列并加入了新的起始密码子ATG，序列分析表明，与日本报道的人OBcDNA相比，多出一个谷氨酸密码子CAG．将OBcDNA定向克隆至原核表达载体pBV220，构建了重组OB基因表达质粒pBV220-OB．SDS-PAGE证实pBV220-OB在大肠杆菌中可表达分子量为16.7KD的特异蛋白带．
Total RNA was isolated from Chinese Han adipose tissue with RNAzol extraction. The cDNA encoding OB protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR)method using directly the isolated total RNA as the template. The amplified cDNA fragment was inserted into pUC19 plasmid. The nucleotide sequence of the cDNA was determined by Dye Deoxy TM Terminator cycle sequecing and showed that there are start codon ATG but no signal peptides sequence, and another three bases CAG which code glutamic acid compared with that of Japanese. OB cDNA was inserted into prokaryotic expression vector pBV220 and constructed expression plasmid pBV220-OB. SDS-PAGE showed that E. coli DH5 a containing pBV220-OB can express a specific protein, its molecular weight was 16. 7kD.
基金
国家自然科学基金!39400061
关键词
肥胖症
原核表达
基因克隆
载体
构建
Human OB gene, Molecular cloning, Prokaryotic expression
